Abstract:
Neural tube defects (NTDs) are the most common of all serious human birth defects. Studies have identified genetic components that may be linked to increased susceptibility to environmentally-induced NTDs. The SWV and C57BL/J6 mice strains have shown to have a large disparity in sensitivity to environmentally-induced NTDs. When exposed to metals during neurogenesis, the C57BL/J6 has been shown to be more sensitive than the SWV. Due to its complex status of proliferation, differentiation and cell turnover, the developing embryo is highly susceptible to metal-induced alterations in cell cycle and apoptosis. We hypothesize that differential metal-induced impacts within cell cycle and apoptosis pathways contribute to observed differing sensitivities between these two strains. To investigate our hypothesis, a primary in vitro model was established for each strain by isolating murine embryonic fibroblasts (MEFs) at day 14 of gestation. At passage 4-6, MEFs were treated with environmentally relevant concentrations of cadmium (Cd) and methyl mercury (MeHg). A cytotoxicity profile was generated after 24hrs using the neutral red assay. To examine changes in apoptotic pathways, caspase 3/7 and 8 activities were measured after 24hrs and further confirmed by Western blot. To characterize metal induced-alterations within cell cycle pathways: 1) cell cycle kinetics were assessed using flow cytometry (brdu/Hoechst) after 8, 24, and 48hrs, 2) gene transcriptional changes of key cell cycle regulators p21 and cyclin B1 were measured using RTPCR (8 and 24hrs), and 3) metal-induced translational changes of p53 and cdc 20 were quantified using Western blot (24hr only). We observed a dose dependent decrease in cell viability with increasing concentrations of both metals. The SWV exhibited higher sensitivity to Cd, confirmed by a statistically significant shift in cell viability compared to the C57BL/J6. A dose dependent increase of caspase 3/7 activity was observed for both strains across both metals. An increase in caspase 8 activity was also detected at the highest concentrations of MeHg and Cd (C57BL/J6 only). The C57BL/J6 displayed a higher increase in caspase 3 (p£0.05) and caspase 8 (p£0.07) activity compared to the SWV at the highest dose of cadmium. At relatively low cytotoxic levels (£20% reduction in cell viability after 24hrs) both Cd and MeHg significantly repressed cell cycle progression in both SWV and C57BL/J6 MEFs. In summary, in SWV and C57BL/J6 MEFs, we observed dose dependent metal-induced alterations in cell cycle and apoptotic pathways. Furthermore, differences in the magnitude of effect within these pathways were observed between the SWV and C57BL/J6 upon treatment of Cd. These differences may contribute to differing sensitivities observed in these two strains.